• 1.RunproductsofaPCRamplificationon1-2%TBEagarosegel,astwoormorereplicatelanes.
• 2.Cutoff1lane-flankedbyMarkerDNAifdesired,andnotchedtoallowre-orientationwithremainderofgel-andstaininpreferredethidiumbromideconcentration(Iuse50ng/mlfor10min).
• 3.Viewexcisedstainedpieceon254nmUVboxformaximumsenssitivity;notchorstabcorrectband(s)insamplelane.
• 4.Prepare"coresamplers":usingglovesandsterilescissorsandcutoffabout5mmfromthetipofasmanysterileyellowPipettetips(weuseGilsontips)asyouwillneedforsamples.
• 5.Alignstainedmarkedsegmentwithremainderofgel.Use"coresamplers"tostaboutoneormorecoresofagarosefromthecentreofbandsofinterest,usingstabbed/notchedgellaneasreference:astandardgelshouldgiveabout10ulpercore.
• 6.Stainremainderofgel,viewandphotographat254nmtoensurecorrectregionsweresampled.

NOTE:ITISPOSSIBLETOQUICKLYCOREASTAINEDGELDIRECTLYONA305OREVENA254NMUVBOX;HOWEVER,MORETHANAFEWSECONDSOFEXPOSURERESULTSINCROSS-LINKINGANDNOAMPLIFICATION

• 7.UsecoresamplesassubstrateinPCRreactions:Imakeup40ul/reactionofreactionmix,andallow10ulpercore.Simplyaddcoretomix,vortexalittle,spindown,coverwithmineraloil.PCRaccordingtotaste(notinhibitedbypresenceofalittlebromophenolblueorof50ng/mlethidiumbromide).
• 8.AtendofPCR:ifyouallowthetubestocooldownthereactionmixwillset:2%-oddagarosediluted1/5setsquitewell!ThisisnoproblemforgelrunningasyouthenendthePCRona10min72oCcycle,andloadthesampleintowellsofagelBEFOREsubmergingthegel:samplewillsetinthewellsandnotfloatout.
• 9.IfyouwishtoextractDNA,endat72oCandadd50ulpre-warmedphenol/8-OH-quinolineandvortex,add100ulchloroform/isoamylalcohol(24:1),vortex,spin:agaroseshouldbeinthephenol/CHCl3phase.ALTERNATIVELY:takeoffmineraloilusing50ulCHCl3,takeoutplugofsolidifiedsampleandwashinTE,thenputinto0.5mlEppendorf-typevialwithsomesiliconisedglasswoolatbottom,andasmallneedlehole.PutlittleEppiinbigEppiwithoutalid,andspin6000rpm10min(alaHeeryetal.,1990;TIG6(6):173).Collectfiltrate,cleanupbyphenol/CHCl3andisopropanol/ammoniumacetateppte(1volIP,0.2volof10Mammoniumacetate).

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  Ihavesuccessfullyre-amplifiedaunique500bpbandfromabackgroundofmanybandsupto1.5kbfromaCDNAPCRofcauliflowermosaicvirus35SRNAintotalturnipRNAextract,anda150bpbandfromabackgroundofbandsgoingupto3kbfromanamplificationofArABIdopsistotalgenomicDNAusingthoroughlydegenerateprimers-inthelattercase,toapointwhereitcouldbesequenceddirectly(usingsameprimers)afterasubsequentamplificationafterpurificationfromagelplugasabove.

  Themethodhasadvantagesoverapreviously-describedtoothpickingprocedureinthatacoresampleisgenerallyofdefinedvolume,maybestoredindefinitely,andprovidesmaterialformultiplere-amplifications.

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